1,3-oxathiolane nucleoside analogues and methods for using same

ABSTRACT

The invention relates to 1,3-oxathiolane nucleoside analogues and their use in the treatment of viral infections. More specifically, this invention relates to (-)-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one and pharmaceutically acceptable derivatives and pharmaceutical formulations thereof.

This is a continuation of application Ser. No. 08/190,203 U.S. Pat. No.5,538,975, filed Feb. 1, 1994 entitled 1,3-Oxathiolane NucleosideAnalogues.

The present invention relates to nucleoside analogues and their use inmedicine. More specifically the invention is concerned with1,3-oxathiolane nucleoside analogues, pharmaceutical formulationsthereof and the use thereof in the treatment of viral infections.

The only compound currently approved for the treatment of conditionscaused by HIV is 3'-azido-3'-deoxythymidine (AZT, zidovudine, BW 509U).However, this compound has a significant side-effect liability and thuseither cannot be employed or, once employed, may have to be withdrawn ina significant number of patients. There is in consequence a continuingneed to provide compounds which are effective against HIV but with aconcommitant significantly better therapeutic index.

The compound of formula (I) ##STR1## is a racemic mixture of the twoenantiomers of formulae (I-1) and (I-2): ##STR2##

We have now found that, surprisingly, the (-)-enantiomer of the compoundof formula (I) is much more active than the (+)-enantiomer, althoughboth enantiomers show unexpectedly low cytotoxicity. There is thusprovided in a first aspect of the invention the (-)(or laevorotatory)enantiomer of the compound of formula (I) and pharmaceuticallyacceptable derivatives thereof.

The (-)-enantiomer has the chemical name(-)-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one(hereinafter compound (A)). This enantiomer has the absolutestereochemistry shown in formula (I-1).

Preferably compound (A) is provided substantially free of thecorresponding (+)-enantiomer, that is to say no more than about 5% w/wof the (+)-enantiomer, more preferably no more than about 2%, and mostpreferably less than about 1% w/w is present.

By "a pharmaceutically acceptable derivative" is meant anypharmaceutically acceptable salt, ester, or salt of such ester, ofcompound (A) or any other compound which, upon administration to therecipient, is capable of providing (directly or indirectly) compound (A)or an antivirally active metabolite or residue thereof.

It will be appreciated by those skilled in the art that compound (A) maybe modified to provide pharmaceutically acceptable derivatives thereof,at functional groups in both the base moiety and at the hydroxymethylgroup of the oxathiolane ring. Modification at all such functionalgroups are included within the scope of the invention. However, ofparticular interest are pharmaceutically acceptable derivatives obtainedby modification of the 2-hydroxymethyl group of the oxathiolane ring.

Preferred esters of compound (A) include the compounds in which thehydrogen of the 2-hydroxymethyl group is replaced by an acyl function##STR3## in which the non-carbonyl moiety R of the ester is selectedfrom hydrogen, straight or branched chain alkyl (e.g., methyl, ethyl,n-propyl, t-butyl, n-butyl), alkoxyalkyl (e.g., methoxymethyl), aralkyl(e.g., benzyl), aryloxyalkyl (e.g., phenoxymethyl), aryl (e.g., phenyloptionally substituted by halogen, C₁₋₄ alkyl or C₁₋₄ alkoxy);sulphonate esters such as alkyl- or aralkylsulphonyl (e.g.,methanesulphonyl); amino acid esters (e.g., L-valyl or L-isoleucyl) andmono-, di- or tri-phosphate esters.

With regard to the above described esters, unless otherwise specified,any alkyl moiety present advantageously contains 1 to 16 carbon atoms,particularly 1 to 4 carbon atoms. Any aryl moiety present in such estersadvantageously comprises a phenyl group.

In particular the esters may be a C₁₋₁₆ alkyl ester, an unsubstitutedbenzyl ester or a benzyl ester substituted by at least one halogen(bromine, chlorine, fluorine or iodine), C₁₋₆ alkyl, C₁₋₆ alkoxy, nitroor trifluoromethyl groups.

Pharmaceutically acceptable salts of the compound (A) include thosederived from pharmaceutically acceptable inorganic and organic acids andbases. Examples of suitable acids include hydrochloric, hydrobromic,sulphuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic,lactic, salicylic, succinic, toluene-p-sulphonic, tartaric, acetic,citric, methanesulphonic, formic, benzoic, malonic,naphthalene-2-sulphonic and benzenesulphonic acids. Other acids such asoxalic, while not in themselves pharmaceutically acceptable, may beuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and NR₄ +(where R is C₁₋₄ alkyl) salts.

References hereinafter to a compound according to the invention includeboth the compound (A) and its pharmaceutically acceptable derivatives.

The compounds of the invention either themselves possess antiviralactivity and/or are metabolizable to such compounds. In particular thesecompounds are effective in inhibiting the replication of retroviruses,including human retroviruses such as human immunodeficiency viruses(HIV's), the causative agents of AIDS.

The compounds of the invention are also useful in the treatment ofanimals including man infected with the hepatitis B virus (HBV).

There is thus provided as a further aspect of the invention compound (A)or a pharmaceutically acceptable derivative thereof for use as an activetherapeutic agent in particular as an antiviral agent, for example inthe treatment of retroviral infections or HBV infections.

In a further or alternative aspect there is provided a method for thetreatment of a viral infection, in particular an infection caused by HBVor a retrovirus such as HIV, in a mammal including man comprisingadministration of an effective amount of compound (A) or apharmaceutically acceptable derivative thereof.

There is also provided in a further or alternative aspect use ofcompound (A) or a pharmaceutically acceptable derivative thereof for themanufacture of a medicament for the treatment of a viral infection.

The compounds of the invention are also useful in the treatment of AIDSrelated conditions such as AIDS-related complex (ARC), progressivegeneralized lymphadenopathy (PGL), AIDS-related neurological conditions(such a dementia or tropical paraparesis), anti-HIV antibody positiveand HIV-positive conditions. Kaposi's sarcoma, thrombocytopenia purpureaand associated opportunistic infections for example pneumocystiscarinii.

The compounds of the invention are also useful in the prevention ofprogression to clinical illness of individuals who are anti-HIV antibodyor HIV-antigen positive and in prophylaxis following exposure to HIV.

The compound (A) or pharmaceutically acceptable derivatives thereof mayalso be used for the prevention of viral contamination of physiologicalfluids such as blood or semen in vitro.

The compounds of the invention are also useful in the treatment ofanimals including man infected with the hepatitis B virus.

It will be appreciated by those skilled in the art that reference hereinto treatment extends to prophylaxis as well as the treatment ofestablished infections or symptoms.

It will be further appreciated that the amount of a compound of theinvention required for use in treatment will vary not only with theparticular compound selected but also with the route of administration,the nature of the condition being treated and the age and condition ofthe patient and will be ultimately at the discretion of the attendantphysician or veterinarian. In general however a suitable dose will be inthe range of from about 0.1 to about 750 mg/kg of bodyweight per daypreferably in the range of 0.5 to 60 mg/kg/day, most preferably in therange of 1 to 20 mg/kg/day.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example as two,three, four or more sub-doses per day.

The compound is conveniently administered in unit dosage form; forexample containing 10 to 1500 mg, conveniently 20 to 1000 mg, mostconveniently 50 to 700 mg of active ingredient per unit dosage form.

Ideally the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 1 to about 75μM, preferably about 2 to 50 μM, most preferably about 3 to about 30 μM.This may be achieved, for example, by the intravenous injection of a 0.1to 5% solution of the active ingredient, optionally in saline, or orallyadministered as a bolus containing about 1 to about 100 mg of the activeingredient. Desirable blood levels may be maintained by a continuousinfusion to provide about 0.01 to about 5.0 mg/kg/hour or byintermittent infusions containing about 0.4 to about 15 mg/kg of theactive ingredient.

While it is possible that, for use in therapy, a compound of theinvention may be administered as the raw chemical it is preferable topresent the active ingredient as a pharmaceutical formulation.

The invention thus further provides a pharmaceutically formulationcomprising compound (A) or a pharmaceutically acceptable derivativethereof together with one or more pharmaceutically acceptable carrierstherefor and, optionally, other therapeutic and/or prophylacticingredients. The carrier(s) must be `acceptable` in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

Pharmaceutical formulations include those suitable for oral, rectal,nasal, topical (including buccal and sub-lingual), vaginal or parenteral(including intramuscular, sub-cutaneous and intravenous) administrationor in a form suitable for administration by inhalation or insufflation.The formulations may, where appropriate, be conveniently presented indiscrete dosage units and may be prepared by any of the methods wellknown in the art of pharmacy. All methods include the step of bringinginto association the active compound with liquid carriers or finelydivided solid carriers or both and then, if necessary, shaping theproduct into the desired formulation.

Pharmaceutical formulations suitable for oral administration mayconveniently be presented as discrete units such as capsules, cachets ortables each containing a predetermined amount of the active ingredient;as a powder or granules; as a solution, a suspension or as an emulsion.The active ingredient may also be presented as a bolus, electuary orpaste. Tablets and capsules for oral administration may containconventional excipients such as binding agents, fillers, lubricants,disintegrants, or wetting agents. The tablets may be coated according tomethods well known in the art. Oral liquid preparations may be in theform of, for example, aqueous or oily suspensions, solutions, emulsions,syrups or elixirs, or may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles (which may includeedible oils), or preservatives.

The compounds according to the invention may also be formulated forparenteral administration (e.g., by injection, for example bolusinjection or continuous infusion) and may be presented in unit dose formin ampoules, pre-filled syringes, small volume infusion or in multi-dosecontainers with an added preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation orsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

For topical administration to the epidermis the compounds according tothe invention may be formulated as ointments, creams or lotions, or as atransdermal patch. Ointments and creams may, for example, be formulatedwith an aqueous or oily base with the addition of suitable thickeningand/or gelling agents. Lotions may be formulated with an aqueous or oilybase and will in general also contain one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents, thickeningagents, or coloring agents.

Formulations suitable for topical administration in the mouth includelozenges comprising active ingredient in a flavored base, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert base such as gelatin and glycerin or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the active compound with thesoftened or melted carrier(s) followed by chilling and shaping inmoulds.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

For intra-nasal administration the compounds of the invention may beused as a liquid spray or dispersible powder or in the form of drops.

Drops may be formulated with an aqueous or non-aqueous base alsocomprising one or more dispersing agents, solubilizing agents orsuspending agents. Liquid sprays are conveniently delivered frompressurized packs.

For administration by inhalation the compounds according to theinvention are conveniently delivered from an insufflator, nebulizer or apressurized pack or other convenient means of delivering an aerosolspray. Pressurized packs may comprise a suitable propellant such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecompounds according to the invention may take the form of a dry powdercomposition, for example a powder mix of the compound and a suitablepowder base such as lactose or starch. The powder composition may bepresented in unit dosage form in, for example, capsules or cartridges ore.g., gelatin or blister packs from which the powder may be administeredwith the aid of an inhalator or insufflator.

When desired the above described formulations adapted to give sustainedrelease of the active ingredient may be employed.

The pharmaceutical compositions according to the invention may alsocontain other active ingredients such as antimicrobial agents, orpreservatives.

The compounds of the invention may also be used in combination withother therapeutic agents for example other antiinfective agents. Inparticular the compounds of the invention may be employed together withknown antiviral agents.

The invention thus provides, in a further aspect, a combinationcomprising the compound (A) or a physiologically acceptable derivativethereof together with another therapeutically active agent, inparticular an antiviral agent.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above together with apharmaceutically acceptable carrier therefor comprise a further aspectof the invention.

Suitable therapeutic agents for use in such combinations include acyclicnucleosides such as acyclovir or ganciclovir, interferons such as alpha,beta or gamma-interferon, renal excretion inhibitors such as probenecid,nucleoside transport inhibitors such as dipyridamole,2',3'-dideoxynucleosides such as AZT, 2',3'-dideoxycytidine,2',3'-dideoxyadenosine, 2',3'-dideoxyinosine, 2',3'-dideoxythymidine,2',3'-dideoxy-2',3'-didehydrothymidine and2',3'-dideoxy-2',3'-didehydrocytidine, immunomodulators such asinterleukin-2 (IL-2) and granulocyte macrophage colony stimulatingfactor (GM-CSF), erythropoietin, ampligen, thymomodulin, thymopentin,foscarnet, ribavirin and inhibitors of HIV banding to CD4 receptorse.g., soluble CD4, CD4 fragments, CD4 hybrid molecules, glycosylationinhibitors such as 2-deoxy-D-glucose, castanospermine and1-deoxynojirimycin.

The individual components of such combinations may be administeredeither sequentially or simultaneously in separate or combinedpharmaceutical formulations.

When the compound (A) or a pharmaceutically acceptable derivativethereof is used in combination with a second therapeutic agent activeagainst the same virus the dose of each compound may be either the sameas or differ from that when the compound is used alone. Appropriatedoses will be readily appreciated by those skilled in the art.

The compound (A) and its pharmaceutically acceptable derivatives may beprepared by any method known in the art for the preparation of compoundsof analogous structure, for example as described in European PatentPublication 0382526 A2.

It will be appreciated by those skilled in the art that for certain ofthe methods described herein below the desired stereochemistry of thecompound (A) may be obtained either by commencing with an optically purestarting material or by resolving the racemic mixture at any convenientstage in the synthesis. In the case of all the processes the opticallypure desired product may be obtained by resolution of the end product ofeach reaction.

In one such process a 1,3-oxathiolane of formula (VIII) ##STR4## whereinthe anomeric group L is a displaceable group, is reacted with anappropriate base. Suitable groups L include --OR where R is an alkylgroup, e.g., a C₁₋₆ alkyl group such as methyl or R is an acyl group,e.g., a C₁₋₆ alkyl group such as acetyl or halogen, for example iodine,bromine or chlorine.

The compound of formula (VIII) is conveniently reacted with5-fluoro-cytosine or an appropriate pyrimidine base precursor thereof(previously silylated with a silylating agent such ashexamethyldisilazane) in a compatible solvent such as methylene chlorideusing a Lewis acid such as titanium tetrachloride,trimethylsilyltriflate, trimethylsilyl iodide (TMSI) or tin (IV)compound such as SnCl₄.

The 1,3-oxathiolanes of formula (VIII) may be prepared for example byreaction of an aldehyde of formula (VII) with a mercaptoacetal offormula (VI) in a compatible organic solvent, such as toluene in thepresence of an acid catalyst for example a Lewis acid such as zincchloride.

    HSCH.sub.2 CH(OC.sub.2 H.sub.5).sub.2                      (VI)

    C.sub.6 H.sub.5 CO.sub.2 CH.sub.2 CHO                      (VII)

The mercaptoacetals of formula (VI) may be prepared by methods known inthe art, for example G. Hesse and I. Jorder, Chem. Ber., 85, pp. 924-932(1952).

The aldehydes of formula (VII) may be prepared by methods known in theart for example E. G. Halloquist and H. Hibbert, Can. J. Research, 8,pp. 9-136 (1933). Conveniently the crude aldehyde (VII) may be purifiedby conversion to the crystalline bisulphite addition adduct andsubsequent reconversion to the free aldehyde.

In a second process the compound (A) is obtained by base interconversionof a compound of formula (IX) ##STR5## where B is a base convertible to5-fluoro-cytosine. Such interconversion may be effected either by simplechemical transformation (e.g. the conversion of uracil base to cytosine)or by an enzymatic conversion using a deoxyribosyl transferase. Suchmethods and conditions for base interconversion are well known in theart of nucleoside chemistry.

In a third process a compound of formula (XI) ##STR6## may be convertedto the compound (A) by conversion of the anomeric NH₂ group to the5-fluoro-cytosine base by methods well known in the nucleoside chemistryart.

Many of the reactions described hereinabove have been extensivelyreported in the context of nucleoside synthesis, for example inNucleoside Analogs--Chemistry, Biology and Medical Applications, R. T.Walker et al., Eds., Plenum Press, New York (1979) at pages 165-192 andT. Ueda, Chemistry of Nucleosides and Nucleotides, Vol I, L. B. TownsendEd., Plenum Press, New York (1988) at pages 165-192, the disclosures ofwhich are incorporated by reference herein.

It will be appreciated that the above reactions may require the use of,or conveniently may be applied to, starting materials having protectedfunctional groups, and deprotection might thus be required as anintermediate or final step to yield the desired compound. Protection anddeprotection of functional groups may be effected using conventionalmeans. Thus, for example, amino groups may be protected by a groupselected from aralkyl (e.g. benzyl), acyl, aryl (e.g. 2,4-dinitrophenyl)or silyl; subsequent removal of the protecting group being effected whendesired by hydrolysis or hydrogenolysis as appropriate using standardconditions. Hydroxyl groups may be protected using any conventionalhydroxyl protecting group, for example, as described in ProtectiveGroups in Organic Chemistry, J. F. W. McOmie, Ed., Plenum Press, NewYork (1973) or T. W. Greene, Protected Groups in Organic Synthesis, JohnWiley and Sons, New York (1981). Examples of suitable hydroxylprotecting groups include groups selected from alkyl (e.g. methyl,t-butyl or methoxymethyl), aralkyl (e.g., benzyl, diphenylmethyl ortriphenylmethyl), heterocyclic groups such as tetrahydropyranyl, acyl(e.g., acetyl or benzoyl) and silyl groups such as trialkylsilyl (e.g.,t-butyldimethylsilyl). The hydroxyl protecting groups may be removed byconventional techniques. Thus, for example, alkyl, silyl, acyl andheterocyclic groups may be removed by solvolysls, e.g., by hydrolysisunder acidic or basic conditions. Aralkyl groups such as triphenylmethylmay similarly be removed by solvolysis, e.g., by hydrolysis under acidicconditions. Aralkyl groups such as benzyl may be cleaved for example bytreatment with BF₃ /etherate and acetic anhydride followed by removal ofacetate groups so formed at an appropriate stage in the synthesis. Silylgroups may also conveniently be removed using a source of fluoride ionssuch as tetra-n-butylammonium fluoride.

In the above processes compound (A) is generally obtained as a mixtureof the cis and trans isomers of which the cis isomer is the compound ofinterest.

These isomers may be separated by physical means, e.g., chromatographyon silica gel or by fractional crystallization, either directly or on asuitable derivative thereof, e.g., acetates (prepared for example withacetic anhydride) followed, after separate, by conversion back to theparent product (e.g., by deacetylation with methanolic ammonia).

Pharmaceutically acceptable salts of the compounds of the invention maybe prepared as described in U.S. Pat. No. 4,383,114, the disclosure ofwhich is incorporated by reference herein. Thus, for example, when it isdesired to prepare an acid addition salt of compound (A) the product ofany of the above procedures may be converted into a salt by treatment ofthe resulting free base with a suitable acid using convention methods.Pharmaceutically acceptable acid addition salts may be prepared byreacting the free base with an appropriate acid optionally in thepresence of a suitable solvent such as an ester (e.g., ethyl acetate) oran alcohol (e.g., methanol, ethanol or isopropanol). Inorganic basicsalts may be prepared by reacting the parent compound with a suitablebase such as an alcohol (e.g., methanol). Pharmaceutically acceptablesalts may also be prepared from other salts, including otherpharmaceutical acceptable salts, of the compound (A) using conventionalmethods.

Compound (A) may be converted into a pharmaceutically acceptablephosphate or other ester by reaction with a phosphorylating agent, suchas POCl₃, or a suitable esterifying agent, such as an acid halide oranhydride, as appropriate. An ester or salt of compound (A) may beconverted to the parent compound for example by hydrolysis.

Resolution of the final product, or an intermediate or starting materialtherefor may be effected by any suitable method known in the art: seefor example E. L. Eliel, Stereochemistry of Carbon Compounds, McGrawHill (1962) and S. H. Wilen, Tables of Resolving Agents.

Thus for example the compound (A) may be obtained by chiral HPLC using asuitable stationary phase for example acetylated β-cyclodextrin orcellulose triacetate and a suitable solvent for example an alcohol suchas ethanol or an aqueous solution of for example triethyl ammoniumacetate. Alternatively the compounds may be resolved by enzyme mediatedenantioselective catabolism with a suitable enzyme such as cytidinedeaminase or selective enzymatic degradation of a suitable derivative a5'-nucleotidase. When resolution is effected enzymatically the enzymemay be employed either in solution or, more conveniently, in immobilizedform. Enzymes may be immobilized by any method known in the art, forexample by adsorption onto a resin such as Eupergit C.

The invention will be further described by the following examples whichare not intended to limit the invention in any way. All temperatures arein degrees Celsius.

Intermediate 1(±)-Cis-2-hydroxymethl-5-(5'-fluorocytosin-1'-yl)-1,3-oxathiolane

(i) 2-Benzoyloxymethyl-5-acetoxy-1,3,,oxathiolane

Benzoyloxyacetaldehyde (216.33 g, 1.32 mol) was dissolved in pyridine(373 ml, 4.61 mol) and 1,4-dithiane-2,5-diol (100.31 g, 0.66 mol) wasadded to the solution. The heterogenous mixture was stirred at 60°-65°C. under nitrogen atmosphere for 1 hour. At the end of the reaction, acomplete solution was obtained. Dichloromethane (650 ml) was added tothe reaction mixture and it was cooled to 0° C. with salt-ice bath.Acetyl chloride (281 ml, 3.95 mol) was added dropwise to the solution at0°-5° C. over 1.5-2 hours. The reaction mixture was stirred at 0°-5° C.for 30 minutes, then it was poured carefully onto a cold (0° C.)solution of saturated sodium bicarbonate. The organic layer wasseparated. The water layer was extracted with dichloromethane (3×200ml). The combined organic layers were washed with saturated sodiumbicarbonate solution (3×200 ml) and brine (200 ml). The solution wasdried over sodium sulfate and concentrated in vacuo. The traces ofpyridine were removed by azeotropic distillation with benzene. 320.79 gcrude product was obtained which was purified by Kugelrohr distillationor filtration through a short silica gel column. [Solvent system:hexane/ethyl acetate (3/1)].

(ii) Cis- and trans-2-benzoyloxymethyl-5-(N₄'-acetyl-5'-fluoro-cytosin-1'-yl)-1,3-oxathiolane

5-Fluorocytosine (4.30 g, 33.3 mmol), hexamethyldisilazane (25 ml) andammonium sulfate (120 mg) were boiled under reflux until the cytosinedissolved (3 hours) and then further refluxed for 2 hours. Thehexamethyldisilazane was evaporated in vacuo and toluene (100 ml) wasadded to the residue to co-evaporate the solvents. The resultingsolution bis(trimethylsilyl)-fluorocytosine in dichloromethane (40 ml)was added under argon to a solution of2-benzoyloxymethyl-5-acetoxy-1,3-oxathiolane (8.537 g, 30.3 mmol) in drydichloromethane (100 ml) and molecular sieves (4A, 2 g) previouslyprepared under argon and cooled at 0° C. for 20 minutes.[(Trifluoromethane-sulfonyl)oxy]trimethyl silane (6 ml, 31 mmol) wasadded to this mixture at 0° C. and the resulting solution was stirred atroom temperature for 2 hours. The filtrate was shaken two times with 300ml of brine and one time with distilled water. The organic layer wasdried over magnesium sulfate, filtered and evaporated to dryness. Thisafforded a crude 5-fluoro-cytosine derivative (10.1 g). R_(f) =0.57(EtOAC:MeOH 9:1).

This residue was acetylated in the next step without furtherpurification. The crude material was dissolved in dry dichloromethane(120 ml) in a 500 ml round bottom flask under argon. Triethylamine (12.7ml, 91.1 mmol) and dimethyl aminopyridine (111 mg, 0.9 mmol) were addedto the solution. The flask was then immersed in an ice bath for 1 hourunder argon. Acetic anhydride (4.3 ml, 45 mmol), distilled over sodiumacetate, was syringed into the cooled flask. The mixture was stirredovernight and then carefully decanted into an erlenmeyer flaskcontaining saturated sodium bicarbonate solution. The product was thenwashed with distilled water followed by brine solution. The methylenechloride portions were dried and evaporated under high vacuum todryness, yielding an acetylated α/β mixture as a colorless foam,weighing 9.6 g after drying. Flash chromatography of this material usingethylacetate: methanol (9:1) afforded 3.1 g, 7.8 mmol (46%) pure trans-and 3.5 g, 8.9 mmol (30%) pure cis- title compounds.

trans-isomer: R_(f) =0.65 in ethyl acetate:methanol 9:1

U.V.: (MeOH) Lambda max: 309 nm

¹ H-NMR δ (ppm in CDCL₃) 8.77 (b, 1H; C₄ '--NH-Ac) 8.06 (m, 2H;aromatic) 7.70 (d, 1H; C₆ '--H, J_(CF) =6.3 Hz) 7.62 (m, 1H; aromatic)7.49 (m, 2H; aromatic) 6.51 (dd, 1H; C₅ --H) 5.91 (dd, 1H; C₂ --H) 4.48(dd, 2H; C₂ --CH₂ OCOC₆ H₅) 3.66 (dd, 1H; C₄ --H) 3.34 (dd, 1H; C₄ --H)2.56 (s, 3H; NH--COCH₃)

cis-isomer: R_(f) =0.58 in ethyl acetate: methanol 9:1

U.V.: (MeOH) Lambda max: 309 nm

¹ H-NMR δ (ppm in CDCl₃) 8.72 (b, 1H; C₄ '--NH-Ac) 8.06 (m, 2H;aromatic) 7.87 (d, 1H; C₆ '--H, J_(CF) =6.2 Hz) 7.60 (m, 1H; aromatic)7.49 (m, 2H; aromatic) 6.32 (dd, 1H; C₅ --H) 5.47 (dd, 1H; C₂ --H) 4.73(dd, 2H; C₂ --CH₂ OCOC₆ H₅) 3.62 (dd, 1H; C₄ --H) 3.19 (dd, 1H; C₄ --H)2.55 (s, 3 H; NH--COCH₃)

(iii) (±)-Cis-hydroxymethyl-5-(5'-fluorocytosin-1'-yl)-1,3-oxathiolane

1.2 g (3.05 mmol) of cis-2-benzoyloxymethyl-5-(N₄'-acetyl-5'-fluorocytosin-1'-yl)-1,3-oxathiolane was stirred in 30 ml ofmethanolic ammonia at 0° C. for 1 hour and then overnight at roomtemperature. The mixture was evaporated under reduced pressure. Theresidue was triturated twice (2×30 ml) with anhydrous ether. The solidresidue was recrystallized in absolute ethanol to give 655 mg (2.64mmol, 87%) of pure cis title product: m.p. 204°-206° C.; R_(f) =0.21 inethylacetate:methanol (9:1). The desired compound was identified by ¹ H,¹³ C-NMR and U.V. Lambda max (H₂ O) 280.9 nm.

cis-isomer:

¹ H-NMR δ (ppm in DMSO-d₆) 8.22 (d, 1H; C₆ '--H, J_(CF) =7.26 Hz) 7.84(d, 2H; C₄ '--NH₂) 6.16 (t, 1H; C₅ --H) 5.43 (t, 1H; C₂ --CH₂ --OH) 5.19(t, 1H; C₂ --H) 3.77 (m, 2H; C₂ --CH₂ OH) 3.35 (dd, 1H; C₄ --H

¹³ C-NMR (DMSO-d₆) ##STR7##

EXAMPLE 1(-)-4-Amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one

(i) (±) Cis-2-hydroxymethyl-5-(5'-fluorocytosin-1'-yl)-1,3-oxathiolanemonophosphate

To a stirred mixture of Intermediate 1 (500 mg, 2.024 mmol) in drytrimethyl phosphate (10 ml) cooled to 0° C., was added dropwisephosphorus oxychloride (1.22 ml, 13.1 mmol). The reaction mixture wasstirred at that temperature for 1 hour and then quenched in ice water.The pH of the cold mixture was adjusted to 3 by the addition of aqueous1N sodium hydroxide, then applied to a charcoal column (5 g, DARCO),which was eluted with water followed by ethanol and aqueous ammonia in a(10:10:1) ratio. Fractions containing crude monophosphate were combinedand evaporated and subsequently was applied to a column containing 15 gof DEAE sephadex A25 (HCO₃ -form). Elution was undertaken with agradient of water (300 ml), 0.1M-NH₄ HCO₃ (300 ml), and 0.2M NH₄ HCO₃(100 ml). Evaporation of appropriate fractions after dilution with water(30 ml) afforded (±)cis-2-hydroxymethyl-5-(5'-fluorocytosin-1'-yl)-1,3-oxathiolanemonophosphate as a white solid R_(f) =0.5 (n.PrOH:NH₄ OH 6:4) yield=612mg, 1.77 mmol, 87.9%. ¹ H NMR δ (ppm in D₂ O). 8.27 (d, 1H, C'₆ --H,J_(H-F) =6.47 Hz), 6.33 (dd, 1H, C₅ --H), 5.47 (t, 1H, C₂ --H), 4.84 (m,2H, C₂ --CH₂ OH), 3.63 (dd, 1H, C₄ H), 3.30 (dd, 1 H, C₄ H). HPLC>99%.

(ii) (+)-Cis-2-hydroxymethyl-5-(5'-fluorocytosin-1'-yl)-1,3-oxathiolane

To a solution Of (±)cis-2-hydroxymethyl-5-(5'-fluorocytosin-1'-yl)-1,3-oxathiolanemonophosphate (100 mg, 0.29 mmol) in 3 ml of glycine buffer solution[glycine (52.6 mg) and magnesium chloride (19 mg) in water (10 ml)], wasadded in one portion 5'-nucleotidase [Sigma, 3.5 mg at 29 unit/mg]. Theresulting mixture was incubated at 37° C. with shaking. The reaction wasmonitored by HPLC [chiral column α-acid glycoprotein (AGP) using 0.2Msodium phosphate as eluant at pH 7 with a flow rate 0.15 ml/min] atdifferent intervals. Only the (+)-enantiomer was observed after 2.5hours. More enzyme (2 mg) was added, and incubation was continued for afurther 3 hours. HPLC analysis clearly showed selective and completehydrolysis of the (+)-enantiomer. The resulting mixture was applied to acolumn of DEAE sephadex A-25 (HCO₃ form). Elution was undertaken withwater (155 ml), followed by 0.1 and 0.2M NH₄ HCO₃ (100 ml each).Appropriate fractions containing the first eluted nucleoside werecombined and concentrated. The remaining solid was purified on a shortcolumn silica using ethyl acetate, methanol (4.5:0.5) as eluant and thenseparated by HPLC (employing the above mentioned conditions). Thisafforded pure (+)-cis-2-hydroxymethyl-5-(5'-fluorocytosin-1'-yl)-1,3-oxathiolane (23 mg, 0.093 mmol, 32%) as a white solid (α)²¹ ₆+123° C. [c, 1.00, MeOH] m.p. 185° C. NMR δ (ppm in DMSO). 8.26 (d, 1H,C'₆ --H, J_(H-F) =5.22 Hz), 7.87 (s, 1H, NH₂, D₂ O exchangeable), 7.63(s, 1H, NH₂, D₂ O exchangeable), 6.20 (dd, 1H, C₅₋ H), 5.48 (t, 1H, C₂H), 5.24 (t, 1H, CH₂ --OH, D₂ O exchange), 3.84 (m, 2H, C₂ --CH₂ OH ),3.50 (dd, 1H, C₄ H), 3.37 (dd, 1H, C₄ H).

(iii) (-)-Cis-2-hydroxymethyl-5-(5'-fluorocytosin-1'-yl)-1,3-oxathiolane

Appropriate fractions from the sephadex column containing the secondeluted nucleoside described in step (ii) were combined and evaporatedunder reduced pressure. The residue was dissolved in 2 ml of water andtreated with alkaline phosphatase (Sigma, 1 ml at 60 units/ml) followedby incubation at 37° C. for 1.5 hours. Solvent was then evaporated andthe residue was purified by column chromatography on silica gel usingEtOAc:MeOH (4:1) as eluent followed by HPLC (separation using the sameconditions mentioned above). This afforded pure(-)-cis-2-hydroxymethyl-5-(5'-fluorocytosin-1'-yl)-1,3-oxathiolane (20mg, 0.081 mmol, 28%) m.p. 190° C. (d) rf=0.21, EtOAc:MeOH (4:1). U.V.:(H₂ O) max: 279.1 nm. ¹ H NMR δ (ppm in DMSO-d₆), 8.25 (d, 1H, C'₆ --H ,J_(HF) =7.26 Hz), 7.88 (b, 1H, C'₄ --NH₂, D₂ O exchangeable), 7.85 (b,1H, C'₄ --NH₂ :D₂ O exchangeable), 5.24 (t, 1H, C₂ --H), 3.83 (m, 2H, C₂--CH₂ --OH), 3.19 (dd, 1H, C₄ --H), 3.15 (dd, 1H, C₄ --H).

Intermediate 2 and Example 2 depict an alternate process for preparingthe compound of formula (A).

Intermediate 21'R,2'S,5'R)-MENTHYL-5R-(5'-FLUOROCYTOSIN-1"-YL)-1,3-OXATHIOLANE-2S-CARBOXYLATE

To a suspension of 5-fluorocytosine (155 mg, 1.2 mmol) in CH₂ Cl₂ (1 mL)at room temperature under an argon atmosphere was added, successively,2,4,6-collidine (0.317 mL, 2.4 mmol) and t-butyldimethylsilyltrifluoromethane-sulfonate (0.551 mL, 2.4 mmol). The resultant mixturewas stirred for 15 minutes and a clear solution was obtained. A solutionof (1'R,2'S,5'R)-menthyl-5R-acetoxy-1,3-oxathiolane-2S-carboxylate (330mg, 1 mmol) in CH₂ Cl₂ (0.5 mL) was introduced, followed byiodotrimethylsilane (0.156 mL, 1.1 mmol). Stirring was continued for 3hours. The mixture was diluted with CH₂ Cl₂ (20 mL) and washedsuccessively with saturated aqueous NaHSO₃, water, brine and then wasconcentrated. The residue was taken up in ether-hexanes (1:1, 10 mL) andsaturated aqueous NaHCO₃ (2 mL) and stirred at room temperature for 15minutes. The aqueous layer was removed and the organic phase wascentrifuged to afford a white solid which was washed with hexanes (3×5mL) and then dried under vacuum. The product(1'R,2'S,5'R)-menthyl-5R-(5"-fluorocytosin-1"-yl)-1,3-oxathiolane-2S-carboxylate(350 mg, 88%) thus obtained contained about 6% of(1'R,2'S,5'R)-menthyl-5S-(5"-fluorocytosin-1"-yl)-1,3-oxathiolane-2S-carboxylate(NMR). This material was recrystallized from MeOH/CH₂ Cl₂ /benzene togive a crystalline product: [α]_(D) ²⁶ +22° (c, 0.19, MeOH); m.p.216°-218° C., ¹ H NMR (CDCl₃) δ0.78 (d, 3H, J=7Hz), 0.91 (t, 6H, J=7.3Hz), 1.00 (m, 2H), 1.39-2.04 (m, 7H), 3.12 (dd, 1H, J=6.6 Hz, 6.1 Hz),3.52 (dd, 1H, J=4.7 Hz, 6.1 Hz), 4.79 (dt, 1H, J=4.4 Hz, 4.3 Hz), 5.46(S, 1H), 5.75 (bs, 1H, exchangeable), 6.42 (5t, 1H, J=5.0 Hz), 8.10 (bs,1H, exchangeable), 8.48 (d, 1H, J=6.6 Hz); ¹³ C NMR (CDCl₃ -DMSO-d₆):δ16.7, 21.2, 22.4, 23.7, 26.6, 31.8, 34.4, 36.6, 40.5, 47.2, 77.1, 79.1,90.8, 126.3 (d, J=33 Hz), 137.1 (d, J=244 Hz), 154.2, 158.3 (d, J=15Hz), 170.1.

EXAMPLE 2 2S-HYDROXYMETHYL-5R-(5'-FLUOROCYTOSIN-1'-YL)-1,3-OXATHIOLANE

To a suspension of lithium aluminum hydride (10 mg, 0.54 mmol) in THF (1mL) at ambient temperature under an argon atmosphere was slowly added asolution of(1'R,2'S,5'R)-menthyl-5R-(5"-fluorocytosin-1"-yl)-1,3-oxathiolane-2S-carboxylate(54 mg, 0.135 mmol) in THF (2 mL). The reaction mixture was allowed tostir for 30 minutes, then quenched with excess methanol (2 mL), followedby the addition of silica gel (3 g). The resultant slurry was subjectedto silica gel column chromatography (EtOAc-Hexane-MeOH, 1:1:1) toprovide a gummy solid which was dried azeotropically with toluene togive 20.7 mg (63%) of a white solid as the product: [α]_(D) ²⁶ +114° (c,0.12, MeOH); ¹ H NMR (DMSO-d₆) δ3.14 (dd, 1H, J=4.3, 11.9 Hz), 3.42 (dd,1H J=5.3, 11.9 Hz), 3.76 (m,2H), 5.18 (m, 1H), 5.42 (t, 1H, J=4.8 Hz),6.14 (m, 1H), 7.59 (br m, 1H, exchangeable), 7.83 (br m, 1Hexchangeable), 8.20 (d, 1H, J=7.66 Hz).

EXAMPLE 3 Biological Activity

(i) Antiviral Activity

Antiviral activity of the compound of Example 1 was determined againstHIV-1 in the following cell lines.

C8166 cells, a human T-lymphoblastoid cell line, infected with HIV-1strain RF.

MT-4 cells, a human T-cell leukaemia cell line, infected with HIV-1strain RF.

Antiviral activity in C8166 cells was determined by inhibition ofsyncytium formation (Tochikura et al Virology, 164, 542-546) and in MT-4cells by inhibition of formazan conversion [Baba et al, Biochem BiophysRes Conmun., 142, pp. 128-134 (1987); Mossman, J. Immun. Meth., 65, pp.55-57 (1983)]. Antiviral activities were also determined by analyzingthe amount of HIV p24 antigen synthesized in the presence and absence ofenantiomers.

The results are shown in Tables 1 and 2 below:

                  TABLE 1                                                         ______________________________________                                                    50% Antiviral Activity (μg/ml)                                                          Inhibition of                                        Assay         Formazan   syncytium formation                                  ______________________________________                                        Cells         MT-4       C8166                                                Virus (HIV-1) HIV-1 RF   HIV-1 RF                                             (+)-enantiomer                                                                              >1         0.04                                                 (-)-enantiomer                                                                              0.14       0.0018                                               Intermediate 1                                                                              0.065      0.013                                                AZT                      0.0038                                               ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        50% Inhibition HIV p24 Synthesis (μg/ml)                                   ______________________________________                                        Cells                  C8166                                                  Virus                  RF                                                     (+)-enantiomer         0.1                                                    (-)-enantiomer         0.0022                                                 Intermediate 1         0.011                                                  AZT                    0.017                                                  ______________________________________                                    

(ii) Cytotoxicity

The cytotoxicities of the compounds of Example 1 and the racemiccompound (Intermediate 1) were determined in two CD4 cell lines: H9 andCEM.

Compounds for test were serially diluted from 100 μg/ml to 0.3 μg/ml(final concentrations) in 96 well microtitre plates. 3.6×10⁴ cells wereinoculated into each well of the plates including drug-free controls.After incubation at 37° C. for 5 days, the viable cell count wasdetermined by removing a sample of cell suspension and counting trypanblue excluding cells in a hemocytometer.

The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        50% Cytotoxicity (μg/ml)                                                   Compound         CEM cells H9 cells                                           ______________________________________                                        (+)-enantiomer   217       334                                                (-)-enantiomer   148       296                                                Intermediate 1   173       232                                                ______________________________________                                    

I claim:
 1. A method for treating a viral infection in a mammalcomprising administering to a mammal in need thereof, an antiviraleffective amount of(-)-Cis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolan-5yl)-(1H)-pyrimidin-2-oneor a pharmaceutically acceptable salt, ester or salt of an esterthereof.
 2. A method for treating a viral infection in a mammalcomprising administering to a mammal in need thereof, an antiviraleffective amount of a mixture of(-)-Cis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl-(1H)-pyrimidin-2-oneor a pharmaceutically acceptable salt, ester or salt of an esterthereof, and(+)-Cis-4-amino-5-fluoro-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-oneor a pharmaceutically acceptable salt, ester or salt of an esterthereof, wherein the (+)-enantiomer is present in an amount of no morethan 5% w/w.
 3. A method of treatment according to claim 2 wherein the(+)-enantiomer is present in an amount of no more than about 2% w/w. 4.A method of treatment according to claim 2 wherein the (+)-enantiomer ispresent in an amount of no more than about 1% w/w.